Process for transforming gramineae and the products thereof

ABSTRACT

A method of producing transformed Gramineae comprising making a wound in a seedling in an area of the seedling containing rapidly dividing cells and inoculating the wound with vir +  Agrobacterium tumefaciens. Also, this same method wherein the vir +  A. tumefaciens contains a vector comprising genetically-engineered T-DNA. There are further provided a transformed pollen grain of a Gramineae, a pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir +  A. tumefaciens, a pollen grain of a Gramineae produced by a plant grown from a seedling infected with vir +  A. tumefaciens containing a vector comprising genetically-engineered T-DNA, a pollen grain of a Gramineae whose cells contain a segment of T-DNA, and Gramineae derived from each of these pollen grains. There are also provided a transformed Gramineae plant, a transformed Gramineae plant derived from a seedling infected with vir +  Agrobacterium tumefaciens, a transformed Gramineae plant derived from a seedling infected with vir +  A. tumefaciens containing a vector comprising genetically-engineered T-DNA and a Gramineae plant whose cells contain a segment of T-DNA. Finally, there are provided transformed Gramineae derived from seedlings infected with vir +  Agrobacterium tumefaciens and transformed Gramineae derived from seedlings infected with vir +  A. tumefaciens containing a vector comprising genetically-engineered T-DNA.

This is a continuation of application Ser. No. 07/067,902, filed Jun.29, 1987, now abandoned, which was a continuation-in-part of applicationSer. No. 06/880,271, filed Jun. 30, 1986, now abandoned.

BACKGROUND OF THE INVENTION

Virulent strains of the soil bacterium Agrobacterium tumefaciens areknown to infect dicotyledonous plants and to elicit a neoplasticresponse in these plants. The tumor-inducing agent in the bacterium is aplasmid that functions by transferring some of its DNA into its hostplant's cells where it is integrated into the chromosomes of the hostplant's cells. This plasmid is called the Ti plasmid, and the virulenceof the various strains of A. tumefaciens is determined in part by thevir region of the Ti plasmid which is responsible for mobilization andtransfer of the T-DNA. The T-DNA section is delimited by two23-base-pair repeats designated right border and left border,respectively. Any genetic information placed between these two bordersequences may be mobilized and delivered to a susceptible host. Onceincorporated into a chromosome, the T-DNA genes behave like normaldominant plant genes. They are stably maintained, expressed and sexuallytransmitted by transformed plants, and they are inherited in normalMendelian fashion.

The lump of plant tumor tissue that grows in an undifferentiated way atthe site of the A. tumefaciens infection is called a crown gall. Cellsof crown gall tumors induced by A. tumefaciens synthesize unusual aminoacids called opines. Different strains of A. tumefaciens direct thesynthesis of different opines by the crown gall cells, and theparticular opine induced is a characteristic of the strain whichinfected the plant. Further, the ability to catabolize the particularopine induced by a given strain is also characteristic of that strain.

Opines are not normally synthesized by A. tumefaciens or by theuninfected host plants. Although it is the T-DNA which codes for theenzymes involved in the synthesis of the opines, the opine synthases,these genes are expressed only in infected plant tissue. This type ofexpression is consistent with the observation that these genes are underthe control of eukaryotic regulatory sequences on the T-DNA.

The most common opines are octopine and nopaline. The opine synthasethat catalyzes the synthesis of octopine is lysopine dehydrogenase, andthe opine synthase that catalyzes the synthesis of nopaline is nopalinedehydrogenase.

When crown gall cells are put into culture, they grow to form a callusculture even in media devoid of the plant hormones that must be added toinduce normal plant cells to grow in culture. A callus culture is adisorganized mass of relatively undifferentiated plant cells. Thisability of crown gall cells to grow in hormone-free media is alsoattributable to the presence of the T-DNA in the transformed host plantcells since genes which direct the synthesis of phyto-hormone are alsoassociated with the T-DNA.

A DNA segment foreign to the A. tumefaciens and to the host plant whichis inserted into the T-DNA by genetic manipulation will also betransferred to host plant's cells by A. tumefaciens. Thus, the Tiplasmid can be used as a vector for the genetic engineering of hostplants. Although, in wild type A. tumefaciens there is only one Tiplasmid per bacterium, in genetically-engineered A. tumefaciens, the virregion and the T-DNA do not have to be carried on the same Ti plasmidfor transfer of the T-DNA to occur. The vir region and the T-DNA can becarried on separate plasmids contained within the same Agrobacterium.

It has generally been assumed that the host range of A. tumefaciens waslimited to the dicotyledons, and that transformation of monocotyledonscould not be effected with this bacterium. Indeed, no one has reportedthe transformation of any member of the monocotyledonous Gramineaefamily by infection with A. tumefaciens.

However, recently, Hooykaas-Van Slogteren et al., in Nature, 311, 763(1984), reported the production of small swellings at wound sitesinfected with A. tumefaciens on monocotyledons of the Liliaceae andAmaryllidaceae families. Opines were detected in plant cells taken fromthe wound sites of the infected plants.

Also, Hernalsteens et al. reported in The EMBO Journal, 3, 3039 (1984)that cultured stem fragments of the monocotyledon Asparagus officinalis,a member of the family Liliaceae, infected with A. tumefaciens strainC58 developed tumorous proliferations. One of these tumorousproliferations could be propagated on hormone-free medium, and opineswere detected in the established callus culture derived from thistumorous proliferation.

In 1982, Anne C. F. Graves reported in her Ph.D. Dissertation entitled"Some Tumorigenic Activities of Agrobacterium Tumefaciens (Smith andTown) Conn." (Bowling Green State University) that irregular masses oftissue developed on gladiolus discs in response to inoculations with A.tumefaciens C58N and B6. These masses of tissue appeared to be the sameas, and have cellular morphology similar to, those that develop onpotato tuber discs. Gladiolus is a member of the monocotyledonousIridaceae family. A compound that co-migrated with the octopine standardduring electrophoresis was found in the proliferations on the gladiolusdiscs that were induced by strain B6, and one that migrated just behindthe octopine standard occurred in those induced by C58N. Also, octopinedehydrogenase was found in extracts of the cellular proliferationsinduced by A. tumefaciens B6 but not in those induced by A. tumefaciensC58N.

Dr. Graves also described the response of certain other monocots toinoculation with A. tumefaciens. No cellular proliferation was observedon ginger root rhizome discs, and the results with tulip bulb discs wereinconclusive. Cellular proliferations on discs of the rhizomes ofcattail and skunk cabbage were limited to several layers of clear cellsat the ends of vascular bundles in the early spring.

DeCleene and DeLey in The Botanical Review, 42, 389 (1976) reported theresults of an extensive study of the plant host range of A. tumefaciens.Their article teaches that monocots of the orders Liliales and Aralesare susceptible to infection with A. tumefaciens but that monocotyledonsin general are unsusceptible to A. tumefaciens infection. In particular,their article reports that the Gramineae tested were not susceptible toinfection with A. tumefaciens. Susceptibility to A. tumefaciensinfection was determined by whether a swelling or tumor developed at thewound site.

Lorz et al. in Mol. Gen. Genet., 199, 178 (1985), Fromm et al in Nature,319, 791 (1986) and Portrykus et al. in Mol. Gen. Genet, 199, 183 (1985)have reported the transformation of Gramineae by direct gene transfer toprotoplasts. Protoplasts are plant cells from which the cell wall hasbeen removed by digestion with enzymes. Lorz et al. transformedprotoplasts of Triticum monococcum using a DNA construct containing thenopaline synthase promotor and the polyadenylation regulatory signal ofthe octopine synthase gene. Fromm et al discloses that theelectroporation-mediated transfer of plasmid pCaMVNEO (comprising thecauliflower mosaic virus 35 S promoter, the neomycin phosphotransferaseII gene from the transposon Tn5 and the nopaline synthase 3' region)into maize protoplasts results in stably-transformed maize cells thatare resistant to kanamycin.

To obtain transformed plants from the transformed cells generated usingeither the infection techniques of Hooykaas-Van Slogteren et al.,Hernalsteens et al., Graves and DeCleene and DeLey or the direct genetransfer techniques of Lorz et al., Fromm et al. and Portrykus et al.,the plants would have to be regenerated from protoplasts or single cellcultures. However, no one has yet been able to regenerate plants fromprotoplasts or single cell cultures of the Gramineae. Indeed, no meansof producing transformed plants or other transformed differentiatedorgans or tissues of the Gramineae is currently known, and no method yetexists for transforming the Gramineae in a manner which allows for theexpression of exogenous DNA in agriculturally important forms or partsof the Gramineae such as seeds, pollen, ears or plants.

Finally, PCT International Publication No. WO 86/00931 (Simpson et al.)published Feb. 13, 1986, teaches in vivo methods for transforming andregenerating intact plants. This patent application discloses that themethods of the invention can be used for the transformation of any plantthat forms a shooty tumor following infection with an A. tumefaciensshooty mutant strain. However, as noted above, the Gramineae are notknown to produce tumors or even swellings in response to inoculationwith A. tumefaciens. In the practice of the present invention, notumors, swellings or cellular proliferations of any kind have beenobserved on the Gramineae in response to inoculation with A.tumefaciens.

SUMMARY OF THE INVENTION

According to the present invention, there is now provided a method ofproducing transformed Gramineae comprising making a wound in a seedlingin an area of the seedling containing rapidly dividing cells andinoculating the wound with vir⁺ A. tumefaciens. In the preferredpractice of the invention, the wound is made in an area of the seedlingwhich gives rise to the germ line cells. Also, preferred is the use ofvir⁺ A. tumefaciens which contains a vector comprisinggenetically-engineered T-DNA.

According to another aspect of the invention, there are provided: (1)transformed pollen grains; (2) a transformed pollen grain produced by aplant grown from a seedling infected with vir⁺ A. tumefaciens; (3) atransformed pollen grain produced by a plant grown from a seedlinginfected with vir⁺ A. tumefaciens which contains a vector comprisinggenetically-engineered T-DNA; (4) a pollen grain whose cells contain atleast a segment of T-DNA; and (5) Gramineae derived from each of thesefour pollen grains. In addition, there are provided: (1) a transformedGramineae plant; (2) a transformed Gramineae plant derived from aseedling infected with vir⁺ A. tumefaciens; (3) a transformed Gramineaeplant derived from a seedling infected with vir⁺ A. tumefaciens whichcontains a vector comprising genetically-engineered T-DNA; and (4) aGramineae plant whose cells contain a segment of T-DNA. Finally, thereare provided transformed Gramineae derived from seedlings infected withvir⁺ A. tumefaciens and transformed Gramineae derived from seedlingsinfected with vir⁺ A. tumefaciens which contains a vector comprisinggenetically-engineered T-DNA.

The invention is clearly useful since it provides, for the first time, amethod for transforming Gramineae which results in the production oftransformed differentiated organs and tissue such as leaves, plants andpollen. Thus, the invention provides, for the first time, a method oftransforming Gramineae which allows for the expression of exogenous DNAin agriculturally important forms or parts of the Gramineae. Many of theGramineae (such as corn, oats, rye, barley, sorghum, rice and wheat)are, of course, commercially important food sources for humans and otheranimals, and the invention allows for the development of strains ofGramineae having altered or superior traits, such as higher yieldingstrains and strains having resistance to herbicides or betternutritional value, by providing a means whereby exogenous DNA coding forsuch traits can be incorporated into the Gramineae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-18 show a corn seedling which is ninety-six hours old. FIG. 1Ais the side view of the seedling, and FIG. 1B is the front view of thesame seedling.

FIG. 2A is a drawing of developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of yellow Iochief corn seedlingsinoculated with A. tumefaciens strain B6 with a reaction mediumcontaining reagents which allow for the detection of lysopinedehydrogenase enzyme activity (lysopine dehydrogenase reaction medium).Certain controls were also electrophoresed.

FIG. 2B is a drawing of a developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of yellow Iochief corn seedlings with areaction medium containing reagents which allow for the detection ofnopaline dehydrogenase enzyme activity (nopaline dehydrogenase reactionmedium). Certain controls were also electrophoresed.

FIG. 2C is a drawing of a developed paper electrophoretogram which showsthe presence or absence of pyronopaline in various materials.

FIG. 2D is a drawing of a developed paper electrophoretogram. Thematerials electrophoresed were produced by the catabolism by certainstrains of A. tumefaciens of the products produced by incubatingcell-free extracts of yellow Iochief corn seedlings inoculated with A.tumefaciens strain C58 with nopaline dehydrogenase reaction medium.Certain controls were also electrophoresed.

FIG. 3A is a drawing of a developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of single B6-inoculated yellow Iochiefcorn seedlings with lysopine dehydrogenase reaction medium.

FIG. 3B is a drawing of a developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of single yellow Iochief corn seedlingsinoculated with either A. tumefaciens strain A348 or strain 238MX withlysopine dehydrogenase reaction medium.

FIG. 3C is a drawing of a developed paper electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingcell-free extracts of single C58-inoculated yellow Iochief cornseedlings with nopaline dehydrogenase reaction medium.

FIG. 3D is a drawing of a developed paper electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingcell-free extracts of single yellow Iochief corn seedlings inoculatedwith A. tumefaciens strain JK 195 with nopaline dehydrogenase reactionmedium.

FIG. 4A is a drawing of a developed paper electrophoretogram which isthe result of the electrophoresis of the cell-free sonicates of A.tumefaciens strains B6 and C58.

FIG. 4B is a drawing of a developed paper electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingthe cell-free sonicates of A. tumefaciens strains B6 and C58 for fourhours with an appropriate reaction medium. Also electrophoresed were thereaction media alone.

FIG. 4C is a drawing of a developed paper electrophoretogram which showsthe results of the electrophoresis of the product produced by incubatingthe cell-free extracts of single uninfected seedlings of the yellowIochief strain of corn for four hours with lysopine dehydrogenasereaction medium or with nopaline dehydrogenase reaction medium.

FIG. 5A is a drawing of a developed electrophoretogram which is theresult of the electrophoresis of the products produced by incubating thecell-free extracts of single embryonic leaves from plants grown fromC58-inoculated yellow Iochief corn seedlings with nopaline dehydrogenasereaction medium.

FIG. 5B is a drawing of a corn leaf of meristematic origin showing theareas dissected for assay for nopaline dehydrogenase activity.

FIG. 5C is a drawing of a developed electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingthe cell-free extracts of the dissected sections of four meristematicleaves from four separate plants grown from C58-inoculated yellowIochief corn seedlings with nopaline dehydrogenase reaction medium.

FIG. 5D is a drawing of a developed electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingcell-free extracts of pollen from individual plants grown fromC58-inoculated yellow Iochief corn seedlings with nopaline dehydrogenasereaction medium.

FIG. 6 is a drawing of a developed electrophoretogram which is theresult of the electrophoresis of the products produced by incubating thecell-free extracts of single seedlings of yellow Iochief corn inoculatedwith A. tumefaciens strain CA19 with lysopine dehydrogenase reactionmedium.

FIG. 7 is a drawing of a developed electrophoretogram which shows theresults of the electrophoresis of the products produced by incubatingthe cell-free extracts of single C58-inoculated seedlings of strain PA91corn with nopaline dehydrogenase reaction medium.

FIG. 8 is a restriction site and function map of plasmid pCEL30.

FIG. 9 is a restriction site and function map of plasmid pCEL44.

FIG. 10 is a drawing of developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of single B6-inoculated rye seedlings withlysopine dehydrogenase reaction medium.

FIG. 11 is a drawing of a developed paper electrophoretogram which showsthe results of the electrophoresis of the products produced byincubating cell-free extracts of single B6-inoculated barley seedlingswith lysopine dehydrogenase reaction medium.

FIG. 12 is a drawing of a developed paper electrophoretogram showing theresults of the electrophoresis of the products produced by incubatingcell-free extracts of single C58-inoculated oat seedlings with nopalinedehydrogenase reaction medium.

FIG. 13 is a drawing of a developed electrophoretogram which is theresult of the electrophoresis of the products produced by incubating thecell-free extracts of single C58-inoculated wheat seedlings withnopaline dehydrogenase reaction medium and with lysopine dehydrogenasereaction medium.

FIG. 14 is a drawing of a developed electrophoretogram showing assaysfor enzymatic activity in the upper leaves of transformed plants.

In all of the drawings where they are used, the designations "O" and"OCT" mean octopine, and the designations "N" or "NOP" mean nopaline.These designations are used on the drawings to refer to the lane of theelectrophoretogram containing the synthetic octopine or nopalinestandard or to show the location of spot formed by the syntheticoctopine or nopaline standard after electrophoresis.

In some of the drawings there are spots which do not co-migrate witheither the synthetic octopine or nopaline standards. These spots areformed by unreacted reactants in reaction media or are formed bynaturally-occurring materials found in the cell-free extracts of thecorn seedlings.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

According to the invention, there is provided a method of producingtransformed Gramineae comprising making a wound in a seedling in an areaof the seedling containing rapidly dividing cells and inoculating thewound with vir⁺ A. tumefaciens. The term Gramineae, as used herein, ismeant to include all of the forms and parts of the Gramineae, includingplants, seeds, seedlings, pollen, kernels, ears, leaves, stalks andembryos. Similarly, the terms "corn", "oats", "wheat," "rye" and"barley" are meant to include all forms and parts of the corn, oats,wheat, rye or barley. "Transformed" is used herein to mean geneticallymodified by the incorporation of exogenous DNA into cells. "ExogenousDNA" is DNA not normally found in the strain of Gramineae which is to betransformed. Exogenous DNA may be obtained from prokaryotic oreukaryotic sources, including strains of Gramineae other than the one tobe transformed.

In practicing the method of the invention, the strain of Gramineae to betransformed is sterilized and is then germinated until the radicle(primary root) and stem emerge from the seed. This occurs after aboutfour days of germination, but this time may be shortened by firstsoaking the seeds.

The wound is preferably made in an area of rapidly dividing cells whichgives rise to germ line cells. After making the wound, the seedling isinoculated by dripping a solution of vir⁺ A. tumefaciens into the wound.Vir⁺ A. tumefaciens are bacteria which are capable of mobilizing andtransferring T-DNA into host plant cells, and an A. tumefaciens carryinga plasmid, whether natural or engineered, coding for these functions isvir⁺. Thus, a strain of A. tumefaciens that carries a wild-type Tiplasmid is vir⁺ and can be used in the present invention. Many suchstrains are known and are publicly available. See e.g., American TypeCulture Collection Catalogue (ATCC) of Strain I, p. 66 (15th edition,1982). The vir⁺ region of a wild type Ti plasmid can be used to mobilizeand transfer T-DNA on the same Ti plasmid or to deliver T-DNA on anotherplasmid contained in the same bacterium. In addition, the mobilizationand transfer functions can be supplied by helper plasmids. Such helperplasmids have been described by Ditta et al. in PNAS, 77, 7347 (1980)and by Bagdasarian et al. in Gene, 16, 237 (1981). Thus, a strain of A.tumefaciens that carries a helper plasmid is also vir⁺. Finally, themobilization and transfer functions may be coded on the same engineeredplasmid which contains the T-DNA, and bacteria containing such a plasmidare also vir⁺.

The T-DNA transferred by the vir⁺ A. tumefaciens may be native T-DNA ormay preferably be genetically-engineered T-DNA. Genetically-engineeredT-DNA is a DNA construct comprising T-DNA border sequences, aheterologous gene and a transcription unit connected in operable order.Methods of preparing such constructs are known in the art.

A heterologous gene is a gene which is not normally found in the T-DNAand which is also not normally found in the DNA of the strain ofGramineae which is to be transformed. Heterologous genes may be isolatedfrom prokaryotic and eukaryotic sources, including strains of Gramineaeother than the one to be transformed. Of particular interest are thoseheterologous genes which confer agronomically significant traits onplants containing them.

The heterologous gene is flanked by a transcription unit containing,e.g., promotors and terminators, which allow for expression of theheterologous gene in the strain of Gramineae to be transformed. Theheterologous gene-transcription-unit construct is flanked by the bordersequences. Any T-DNA border sequence, native or synthesized, can be usedto flank the heterologous-gene-transcription-unit construct as long asthe border sequence functions to integrate the heterologous gene intothe cell genome of the strain of Gramineae to be transformed. Thegenetically-engineered T-DNA is linked to a DNA fragment containing areplicon that is functional in Agrobacterium to form a vector.

After the seedlings are inoculated with the vir⁺ A. tumefaciens, theyare incubated until transformation has taken place at which time theseedlings are planted and allowed to grow at least until such time asthey have produced pollen. It is interesting to note that using themethod of the invention, no tumorous growth of any kind, including crowngalls, calli or tumorous overgrowths, has been observed, even on theoriginal inoculated seedling.

By inoculating the seedlings in the preferred area, transformation ofpollen has been achieved. The resulting transformed pollen can be usedto fertilize transformed and untransformed plants. The embryos can beexcised from the resultant progeny ears and grown to produce transformedplants. Alternatively, of course, the plants resulting from this matingcan be allowed to produce seeds which are, in turn, used to growtransformed whole plants which produce another crop of seeds. Thus,future-generations can be derived from the original transformed seedlingby sexual reproduction. Those skilled in the art will recognize thatvarious and numerous progeny carrying the trait coded for by theexogenous DNA can be produced using known breeding techniques.

TRANSFORMATION OF CORN EXAMPLE I A. Preparation of Bacteria

A single colony of the A. tumefaciens strain B6 was inoculated into ayeast extract broth (YEB) containing 0.1% yeast extract, 0.8% nutrientbroth and 0.5% sucrose dissolved in water. The yeast extract andnutrient broth were purchased from Difco Laboratories, Detroit, Mich.The sucrose was purchased from either Fisher Scientific, Detroit, Mich.,or Sigma, St. Louis, Mo. The bacteria were incubated in the YEB for 48hours at 27° C. in a shaking water bath or until such time as they hadreached a final concentration of 3.8×10⁹ cells per milliliter.

The B6 strain is a standard wild type strain of A. tumefaciens. It isvirulent (vir⁺), and it codes for the production of lysopinedehydrogenase in suitable plant hosts. It was obtained from James andBarbara Lippincott, Northwestern University, Evanston, Ill. Some of itsproperties have been described in Stonier, J. Bact., 79, 889 (1960). TheB6 strain is also on deposit at the American Type Culture Collection(ATCC), Rockville, Md., and it has been given accession number 23308.

B. Preparation of Corn

Seeds of the inbred yellow Iochief strain of corn were obtained from TheAndersons, Maumee, Ohio, or from Botzum, 43 East Market, Akron, Ohio.This strain is a standard strain which is available commercially.

The yellow Iochief seeds were sterilized using the following procedure.The seeds were first immersed for two minutes in a solution containing 7parts 95% ethanol and 2.5 parts distilled water. Next, the seeds wereincubated for five minutes in a solution of 0.5% (weight/volume) HgCl₂in distilled water. Next, the seeds were washed for a total of thirtyminutes in a solution of 15% (volume/volume) Clorox (Clorox is anaqueous solution containing 5.25% sodium hypochlorite) and 0.1%(volume/volume) of a liquid dishwashing detergent such as Palmolive oranother suitable wetting agent in distilled water. Finally, the seedswere washed five times in sterile double distilled water.

The sterilized seeds were placed embryo side up on sterile moistenedWhatman No. 3 filter paper contained in a sterile Petri dish. The seedswere incubated in the covered Petri dishes in constant darkness at 25°C. for four days. The filter paper was kept moist throughout theincubation period.

C. Inoculation of Seedlings with A. tumefaciens

The two primary areas of rapidly dividing cells in the corn seedling arethe root cap and the area extending from the base of the scutellar nodethrough the mesocotyl slightly beyond the coleoptile node, the locationsof which are depicted in FIGS. 1A and 1B. The mesocotyl is the areabetween the scutellar node and the coleoptile node.

In the preferred practice of the invention, the wound is made in thearea extending from the base of the scutellar node through and slightlybeyond the coleoptile node because, included within the region, aretissues which give rise to germ line cells. In particular, found in thisregion is tissue which gives rise to the axillary primordia which, inturn, gives rise to tillers and to the ears (female reproductiveorgans). Also, located in this region is the apical meristem which givesrise to the tassle which, in turn, gives rise to the pollen (the malereproductive organs). By inoculating the seedling in this preferredarea, transformation of germ line cells has been obtained.

Accordingly, a total of four wounds were made on the surface of each ofthe germinating corn seedlings prepared in part C of this example in anarea which extends from the base of the scutellar node through andslightly beyond the coleoptile node. The wounds were made by visualizinga line which bisects this area longitudinally (the midline) as theseedling is viewed from the front as in FIG. 1B. The cuts were madeperpendicular to the midline, from the midline to the outside edge ofthe seedling and from the front surface of the seedling, when theseedling is viewed as in FIG. 1B, through all of the tissue in the areawhere the cut is made. Thus, when a cut is made in the area of thescutellar node, the cut is made from the front surface through all ofthe tissue into the scutellum, and, when a cut is made in the mesocotyl,the cut is made from the front surface completely through all of themesocotyl tissue. The four wounds are indicated by the numeral 1 in FIG.IB.

The wounds were inoculated by dripping a total of 100 ul of a 10⁹cells/milliliter suspension of A. tumefaciens strain B6 in YEB, culturedas described above in part A, onto the four wounds. As a control, someseedlings were inoculated with 0.9% NaCl (saline). After receiving theinoculum, the seedlings were placed embryo side up on a layer ofBactoagar contained in a Petri dish, at 5 seedlings per dish. The Petridish contained 20 milliliters of sterile Bactoagar (purchased from DifcoLaboratories) at a concentration of 20 grams/liter in distilled water.The covered Petri dishes were incubated at 27° C. in constant darknessfor an additional 7-14 days.

D. Assays

1. Assay For Enzyme Activity in Seedlings: At the end of the 7-14 dayincubation period, the seedlings were homogenized in a 0.1M Tris-HClbuffer, 8.0, containing 0.5M sucrose, 0.1% (weight/volume) ascorbic acidand 0.1% (weight/volume) cysteine-HCl, using a Wheaton tissue grinder,until the homogenate had a homogeneous consistency. Since the seedlingswere grown in the dark, pigment formation was retarded, and cell wallsremained unusually soft. Thus, the cells of the seedlings broke openeasily. Next, the homogenates were spun in a Fisher Microfuge at 13,000xg for two minutes to obtain cell-free extracts.

A portion of the cell-free extract was added to an equal volume of areaction medium designed to detect lysopine dehydrogenase activity. Thisreaction medium consisted of 30 mM L-arginine, 75 mM pyruvate and 20 mMNADH dissolved in 0.2M sodium phosphate buffer, pH 7.0. The enzymereaction was allowed to proceed at room temperature for the timesindicated below.

The products of the enzyme reaction were separated electrophoreticallyon Whatman 3MM paper. At the start of the enzyme reaction period (timezero), a 5 ul sample of the reaction mixture was spotted at the anodalsite on the paper and dried. After 15 hours of reaction, another 5 ulsample of the reaction mixture was spotted on the paper and dried.Finally, a 5 ul sample of a 100 ug/ml solution of synthetic octopinepurchased from Calbiochem, Division of American Hoescht, La Jolla,Calif., and a 5 ul sample of 100 ug/ml solution of synthetic nopalinepurchased from Sigma, St. Louis, Mo., were spotted on the paper anddried.

Electrophoresis was performed in a formic acid (90.8%)/glacial aceticacid/water (5:15:80, volume/volume) solution, pH 1.8, for 2.5 hours at450 volts. The paper was dried and then stained by dipping it into asolution containing one part 0.02% (weight/volume) phenanthrenequinonein absolute ethanol plus one part 10% (weight/volume) NaOH in 60%(volume/volume) ethanol. After drying, the spots were visualized under along-wave ultraviolet lamp (366 nm).

The results of the electrophoresis of the products produced by addingthe cell-free extracts of B6-inoculated seedlings to lysopinedehydrogenase reaction medium are shown in FIG. 2A. In that figure, lane1 contains a sample of the reaction mixture produced by adding a portionof the cell-free extract of ten B6-inoculated seedlings to an equalvolume of the lysopine dehydrogenase reaction medium at time zero, lane2 contains the product produced by incubating a portion of the cell-freeextract of ten B6-inoculated seedlings with an equal volume of lysopinedehydrogenase reaction medium for fifteen hours, lane 3 contains thesynthetic octopine, lane 4 contains the product produced by adding aportion of the cell-free extract of ten saline-inoculated seedlings toan equal volume of lysopine dehydrogenase reaction medium at time zero,lane 5 contains the product produced by incubating a portion of thecell-free extract of ten saline-inoculated seedlings with an equalvolume of lysopine dehydrogenase reaction medium for fifteen hours, andlane 6 contains the synthetic nopaline.

The results shown in FIG. 2A demonstrate that octopine production iscaused by cell-free extracts of B6-inoculated corn seedlings, lane 2,but that no such production occurs in the saline control, lane 5.Furthermore, the amount of octopine produced, as measured by an increasein phenanthrenequinone fluorescence, increases in proportion to the timeof incubation. While no octopine can be detected at time zero, lane 1,it is clearly present after fifteen hours of incubation, lane 2. Suchresults are in accord with the proposition that the reaction is enzymecatalyzed and that the enzyme extracted from the B6-infected cornseedlings is lysopine dehydrogenase. Since only transformed planttissues are known to express the opine synthase genes, these results arealso in accord with the proposition that the corn seedlings have beentransformed by infection with the vir⁺ A. tumefaciens strain B6.

2. Assay For Substrate Specificity: Lysopine dehydrogenase catalyzes thesynthesis of octopine but not of nopaline. In FIG. 2B the results of theelectrophoresis of the products produced by adding extracts of seedlingsto a reaction medium containing reagents which allow for the detectionof nopaline dehydrogenase enzyme activity are presented. The nopalinedehydrogenase reaction medium consists of 60 mM L-arginine, 60 mMα-ketoglutarate and 16 mM NADH dissolved in 0.2M sodium phosphatebuffer, pH 7.0. The α-ketoglutarate can be used as a substrate only bynopaline dehydrogenase. Lanes 1-6 in FIG. 2B are the same as lanes 1-6of FIG. 2A except that the reaction medium is the nopaline dehydrogenasereaction medium. Thus, FIG. 2B shows that cell-free extracts ofseedlings inoculated with strain B6 cannot use α-ketoglutarate in thecondensation reaction with arginine to produce nopaline. This substratespecificity confirms the identity of the enzyme produced by seedlingstransformed by strain B6 as lysopine dehydrogenase.

3. Transformation Efficiency: To address this issue, assays of singleseedlings were performed, and the number of cell-free extracts of singleseedlings which produced octopine were determined. The results are shownin FIG. 3A where all ten lanes contain the product produced byincubating the cell-free extracts from single B6-inoculated seedlingswith lysopine dehydrogenase reaction medium for four hours. As shown inFIG. 3A, eight of the ten lanes have a spot which stains withphenanthrenequinone and co-migrates with the octopine standard. Thus,the transformation frequency for this experiment was 80%.

4. Controls: To rule out the possibility that the octopine produced wasa consequence of some secondary and uninteresting event, severaladditional controls were done. As shown in FIG. 4A, electrophoresis ofcell-free sonicates of a suspension of B6 cultured for 48 hours, asdescribed above in part A, failed to disclose any stored octopine (lanes1 and 2). Furthermore, when these sonicates were mixed with lysopinedehydrogenase reaction medium and incubated for four hours, no lysopinedehydrogenase activity could be detected. See FIG. 4B, lanes 1, 2 and 3where the products of this incubation were electrophoresed. Thus,lysopine dehydrogenase activity is not found in bacterial cultures 48hours old. Also, lysopine dehydrogenase reaction medium alone did notcontain any octopine. See FIG. 4B, lane 4, where this reaction mediumalone was electrophoresed. Similarly, no evidence is found for theexistence of this dehydrogenase in uninfected corn seedlings. See FIG.4C which shows an electrophoretogram in which lanes 1-5 contain theproduct produced by incubating cell-free extracts of uninfected singleseedlings for four hours with lysopine dehydrogenase reaction medium.These results confirm that the presence of lysopine dehydrogenaseactivity in the cell-free extracts of B6-inoculated seedlings is due tothe transformation of the seedlings and not to some secondary oruninteresting event.

EXAMPLE II A. Preparation of Bacteria

A single colony of the A. tumefaciens strain C58 was inoculated intoYEB, and the bacteria were incubated as described above in Example I,part A, for strain B6. The C58 strain is a standard wild type strain ofA. tumefaciens. It is vir⁺, and it codes for the production of nopalinedehydrogenase in suitable plant hosts. It was obtained from James andBarbara Lippincott, Northwestern University, Evanston, Ill., or fromClarence Kado, University of California, Department of Plant Pathology,Davis, Calif. It has been described in Depicker et al, Plasmid, 3, 193(1980) and Kao, et al, Molec. Gen. Genet., 188, 425 (1982). Strain C58is also on deposit at the ATCC and has been given accession number33970.

B. Transformation of Corn

Seeds of the inbred yellow Iochief strain of corn were sterilized,germinated, inoculated and further incubated for 7-14 days as describedabove in Example I, parts B and C, except that the corn seedlings wereinoculated with strain C58 rather than strain B6.

C. Assays

1. Assay For Enzyme Activity in Seedlings: At the end of the 7-14 dayincubation period, cell-free extracts of the seedlings were prepared andwere assayed for enzymatic activity as described above in Example I,part D, except that the reaction medium used was the one designed toassay for nopaline dehydrogenase activity. As set forth above in ExampleI, part D, this reaction medium consisted of 60 mM L-arginine, 60 mMα-ketoglutaric acid and 16 mM NADH dissolved in 0.2M sodium phosphatebuffer, pH 7.0.

The results are shown in FIG. 2B. Lane 8 in FIG. 2B contains the productproduced by mixing a portion of the cell-free extract of tenC58-inoculated seedlings with an equal volume of nopaline dehydrogenasereaction medium at time zero, and lane 7 contains the product producedby incubating a portion of the cell-free extract of ten C58-inoculatedseedlings with an equal volume of nopaline dehydrogenase reaction mediumfor fifteen hours. Thus, FIG. 2B shows that nopaline is produced by thecell-free extracts of the C58-inoculated corn seedlings, lane 7, butthat no such production was produced by the saline control, lane 5. Onceagain, the amount of nopaline produced, as measured by an increase inphenanthrenequinone fluorescence, increases in proportion to the time ofincubation. While no nopaline can be detected in a reaction mixture attime zero, lane 8, it is clearly present after fifteen hours ofincubation, lane 7, and such results are in accord with the propositionthat the reaction is enzyme-catalyzed and that the enzyme extracted fromC58-infected seedlings is nopaline dehydrogenase. Since only transformedplant tissues are known to express the opine synthase genes, theseresults are also in accord with the proposition that the corn seedlingshave been transformed by infection with the vir⁺ A. tumefaciens strainC58.

2. Pyronopaline Assay: The synthesis of nopaline by extracts fromseedlings transformed with C58 has been confirmed by criteria other thanelectrophoretic mobility. When nopaline is eluted from a paperchromatogram with water, evaporated by the application of a vacuum toreduce the volume and reacted with an equal volume of hot (100° C.) 2Macetic acid for one hour, pyronopaline is formed. The conversionreaction is diagnostic for nopaline and no other opine.

In FIG. 2C, lane 1 is pyronopaline produced from synthetic nopaline bytreating the synthetic nopaline with hot 2M acetic acid as describedabove, lane 2 is synthetic nopaline (some of which convertsspontaneously to pyronopaline), lane 3 is the product produced byincubating a portion of the cell-free extract from ten C58-inoculatedseedlings with an equal volume of the nopaline dehydrogenase reactionmedium for fifteen hours, and lane 4 is this product treated with hot 2Macetic acid as described above. As can be seen, the product produced byincubating the cell-free extract from C58-inoculated seedlings with thenopaline dehydrogenase reaction mixture, lane 3, is totally converted topyronopaline, lane 4, confirming that C58-inoculated seedlings producenopaline dehydrogenase.

3. Catabolism of Nopaline: Finally, if nopaline is incubated with C58and serves as its sole energy source, the bacteria will grow as thiscompound is degraded. However, B6 grown on a medium containing nopalineas the sole energy source will not break down the nopaline and will notdivide since B6 lacks the specific opine oxidase which is necessary forthe catabolism of nopaline.

An assay based on this principle was used to confirm the nopalineidentity of the product produced by the cell-free extracts ofC58-inoculated seedlings. The results of this assay are shown in FIG. 2Dwhere lane 1 contains synthetic nopaline, lane 2 contains the productproduced by incubating a portion of the cell-free extract of tenC58-inoculated seedlings with an equal volume of the nopalinedehydrogenase reaction medium for fifteen hours which has beenelectrophoresed, eluted with water and evaporated, as described above,in connection with the pyronopaline assay, and incubated with strain B6for twenty-four hours, and lane 3 contains the product produced byincubating a portion of the cell-free extract of ten C58-inoculatedseedlings with an equal volume of nopaline dehydrogenase reaction mediumfor fifteen hours which has been electrophoresed, eluted, evaporated andincubated with strain C58 for twenty-four hours. The product produced bythe cell-free extracts of the C58-inoculated seedlings was consumed bystrain C58, lane 3, but was not consumed by strain B6, lane 2,confirming that the product is nopaline.

4. Efficiency of Transformation: The efficiency of the transformation ofinbred yellow Iochief corn using A. tumefaciens strain C58 was alsoinvestigated. The results are shown in FIG. 3C where all ten lanescontain the product produced by incubating cell-free extracts fromsingle C58-inoculated seedlings with nopaline dehydrogenase reactionmedium for six hours. As can be seen, 9 out of 10 seedlings weretransformed. Additional single seedling assays were done using seedlingstransformed with either B6 or C58. Of the 150 total single seedlingassays done with either strain B6 or C58, sixty percent weretransformed.

5. Controls: To rule out the possibility that the nopaline produced wasa consequence of some secondary and uninteresting event, severaladditional controls were done. As shown in FIG. 4A, electrophoresis ofcell-free sonicates of a suspension of C58 cultured for 48 hours, asdescribed above in Example II, part A, failed to disclose any storednopaline, lanes 3 and 4. Furthermore, when these sonicates were mixedwith nopaline dehydrogenase reaction medium and incubated for four hoursat 25° C., no nopaline dehydrogenase activity could be detected. SeeFIG. 4B, lanes 6 and 7 where the products of this incubation wereelectrophoresed. Thus, nopaline dehydrogenase activity was not found inbacterial cultures 48 hours old. Also, nopaline dehydrogenase reactionmedium alone did not contain any nopaline. See FIG. 4B, lane 5, wherethis reaction medium alone was electrophoresed. Similarly, no evidenceis found of this dehydrogenase in uninfected corn seedlings. See FIG. 4Cwhich shows an electrophoretogram in which lanes 6-10 contain theproduct produced by incubating cell-free extracts of uninfected singleseedlings for four hours with nopaline dehydrogenase reaction medium.These results confirm that the presence of nopaline dehydrogenaseactivity in the cell-free extracts of C58-inoculated seedlings is due tothe transformation of the seedlings and not to some secondary anduninteresting event.

EXAMPLE III A. Transformation of Corn

Strain C58 was cultured as described above in Example II, part A, andinbred yellow Iochief corn was sterilized, germinated, inoculated andincubated as described above in Example II, part B. After 7 days ofincubation, the infected seedlings were planted in pots containingpotting soil.

B. Assays

1. Assay For Enzymatic Activity in Leaves of Embryonic Origin: Threeweeks after planting, three leaves of embryonic origin from threeseparate plants were assayed for the presence of nopaline dehydrogenaseactivity. The leaves chosen for the assay were the first leaves from thebase of the plant which had not enlarged. The embryonic leaves arederived from differentiated structures present in the seedlings at thetime of inoculation, but these differentiated structures are not locatedin the inoculated area.

To perform the assay, the three embryonic leaves were individuallyhomogenized in Tris-HCl buffer, centrifuged and assayed for enzymaticactivity as described above in Example II, part C, for the seedlings.The results of the electrophoresis of the product produced by incubatingthe cell-free extracts of the three embryonic leaves with nopalinedehydrogenase reaction medium for twelve hours are shown in FIG. 5Awhere lanes 1, 2 and 3 contain the products of this incubation. As canbe seen from FIG. 5A, none of the cell-free extracts of the embryonicleaves contained nopaline dehydrogenase activity demonstrating thatthese leaves had not been transformed by the inoculation procedure.

2. Assay For Enzymatic Activity in Leaves of Meristematic Origin: Leavesderived from the meristem (all leaves besides embryonic leaves) wereassayed for the presence of nopaline dehydrogenase 7 weeks after theplanting of the seedlings. The meristem is tissue composed of small,rapidly dividing, undifferentiated cells which are capable of dividingto produce organs and other differentiated tissue. Meristematic tissuewhich differentiates into leaves is located in the inoculated area.

To perform the assay, sections were dissected out of themeristem-derived leaves, and the dissected sections from each leaf werehomogenized together in Tris-HCl buffer, centrifuged and assayed forenzymatic activity as described above in Example II, part C, forseedlings. The sections of the leaves which were used for the assay areindicated by the numerals 2 and 3 in FIG. 5B. The section designated bythe numeral 3 was dissected by cutting along lines 6 and 7. Line 7 iscoincident with the midrib 4 of the leaf. Section 3 is located at thebase of the leaf which is normally attached to the plant. The base ofthe leaf is the growing end of the leaf, and this area contains thenewest cells on the leaf. The sections designated by the numeral 2 weredissected by cutting along line 5 which is perpendicular to the midrib4. Sections 2, 2 are at the tip of the leaf, and they contain the oldestcells on the leaf. Each section 2 or 3 constitutes about 1/6 of theleaf's surface area.

The results of the electrophoresis of the product produced by incubatingthe cell-free extracts of these sections of four leaves of meristematicorigin taken from four separate plants with nopaline dehydrogenasereaction medium are shown in FIG. 5C. In FIG. 5C, lane 1 containsnopaline dehydrogenase reaction medium, and lanes 2-5 contain theproducts produced by incubating the cell-free extracts of the leaveswith nopaline dehydrogenase medium for twelve hours. As shown there,cell-free extracts of 3 out of 4 leaves produced nopaline demonstratingthat they contained nopaline dehydrogenase activity. Since these leavesare derived from meristematic tissue by cell division anddifferentiation, these results demonstrate that the cells in theinoculated area of the seedling were able to pass on the ability tosynthesize nopaline dehydrogenase to future generations of corn cells.Thus, these results demonstrate that transformation of cells in the areainoculated and of cells derived from these cells has taken place.

3. Assay For Enzymatic Activity in Pollen: Sixty days after the plantingof the seedlings, samples of the pollen of two plants were individuallyassayed for the presence of nopaline dehydrogenase. To perform theassay, 0.5 to 1.0 milliliter of pollen containing approximately 5-10×10⁵grains of pollen was homogenized in Tris-HCl buffer, centrifuged andassayed for enzymatic activity as described above in Example II, part C,for seedlings. The results of the electrophoresis of the productsproduced by incubating the cell-free extracts of the pollen from the twoplants with nopaline dehydrogenase reaction medium for twelve hours areshown in FIG. 5D where lanes 2 and 3 contain the products of thisincubation, and lane 1 contains nopaline dehydrogenase reaction medium.As can be seen from that figure, pollen from both of the plantscontained nopaline dehydrogenase activity. Since the pollen is derivedfrom the apical meristem by cell division and differentiation, theseresults, like the results for the leaves of meristematic origin above,demonstrate that transformation has taken place.

4. Assay for Enzymatic Activity in Seedlings Derived from TransformedPollen: Pollen from the two transformed plants identified above in partB3 is used to fertilize ears on plants grown from uninfected yellowIochief corn seed. The F₁ seeds produced by the fertilized plants as aresult of this mating are harvested and are germinated and incubated asdescribed above in Example I, parts B and C, except that the seedlingsare not inoculated. After 7-14 days of incubation, the seedlings areassayed for enzymatic activity as described above in Example II, part C.Cell-free extracts of the seedlings are found to produce nopalineshowing that this F₁ generation of seedlings is transformed.

5. Assay for Enzymatic Activity in Leaves of Embryonic Origin Taken fromPlants Derived From Transformed Pollen: Seeds produced by the matingdescribed above in part B4 of this example are harvested and aregerminated and incubated as described above in Example I, parts B and C,except that the seedlings are not inoculated. After 7-14 daysincubation, the seedlings are planted as described above in part A ofthis example. Three weeks after planting, embryonic leaves are assayedfor nopaline dehydrogenase activity as described above in part B1 ofthis example, and cell-free extracts of the embryonic leaves producenopaline showing they are transformed.

EXAMPLE IV A. Transformation of Corn

Yellow Iochief corn was sterilized, germinated, inoculated and furtherincubated for 7-14 days as described above in Example I, Parts B and C,except that separate groups of corn seedlings were inoculated witheither A. tumefaciens strain A348, strain JK 195 or strain 238MX.

The A348 strain carries the broad host range plasmid pTiA6NC from strainA6NC. It is vir⁺, and it codes for the production of lysopinedehydrogenase in suitable plant hosts. Strain A6NC and plasmid pTiA6NCare described by Sciaky et al. in Plasmid, 1, 238 (1977). The A348 usedwas obtained from Eugene Nester, University of Washington, Department ofMicrobiology and Immunology, Seattle, Wash. It was cultured as describedabove in Example I, part A, for strain B6.

Strains JK 195 and 238MX each carry a mutation in the critical virregion and are vir⁻. They cannot, therefore, convey the necessaryportion of the Ti plasmid to their respective hosts. Consequently, plantextracts made from material inoculated with these bacteria would not beexpected to produce any opine when added to the appropriate reactionmedium.

The 238MX is similar in background and source to the A348 strain, buthas the bacterial transposon Tn3 inserted in the vir region rendering itvir⁻. Strain 238MX was obtained from Eugene Nester (address givenabove). It was incubated as described in Example I, part A, for strainB6, and it was selected on YEB containing 100 ug/milliliter ofcarbenicilin.

The JK 195 strain is a vir⁻ mutant derived from C58. It has thebacterial transposon Tn5 inserted in complementation group VI of the virregion. A detailed description of strain JK 195 may be found in Kao etal., Mol. Gen. Genet. 188, 425 (1982) and Lundguist et al., Mol. Gen.Genet., 193, 1 (1984). The JK 195 used was obtained from Clarence Kado(address given above). It was also incubated as described in Example I,part A, and it was selected on YEB containing 50 ug/milliliterrifampicin.

B. Assays

1. Assay For Enzyme Activity in Seedlings: As is shown in FIGS. 3B and3D, cell-free extracts of seedlings inoculated with the 238MX strain orthe JK 195 strain do not produce opines. In FIG. 3B, lanes 6-10 containthe product produced by incubating the cell-free extracts of238MX-inoculated single seedlings with the lysopine dehydrogenasereaction medium for four hours. As can be seen, none of the seedlingswas transformed since no octopine was synthesized by the cell-freeextracts. In FIG. 3D, all ten lanes contain the product produced byincubating the cell-free extracts of JK 195-inoculated single seedlingswith nopaline dehydrogenase reaction medium for six hours. Again, noneof the seedlings was transformed since no nopaline was produced.

However, the A348 strain is competent with respect to transformation. InFIG. 3B, lanes 1-5 contain the product produced by incubating thecell-free extracts of A348-inoculated single seedlings with lysopinedehydrogenase reaction medium for four hours. As can be seen, lysopinedehydrogenase was found in the cell-free extract of one out of fivesingle seedlings inoculated with A348 showing that the seedling wastransformed.

Thus, only those vir⁺ A. tumefaciens strains capable of transferringT-DNA transform corn seedlings. Those which carry mutations in the virregion and which are, therefore, transfer minus do not provoke opinesynthase activity in extracts made from infected plants.

EXAMPLE V

A single colony of the A. tumefaciens strain T37 was inoculated intoYEB, and the bacteria were incubated as described above in Example I,Part A, for strain B6.

The T37 strain is a standard wild type strain of A. tumefaciens. It isvir⁺, and it codes for the production of nopaline dehydrogenase insuitable plant hosts. It was originally obtained from John Kemp,University of Wisconsin, Department of Plant Pathology, Madison, Wis.,and can currently be obtained from Anne C. F. Graves, University ofToledo, Dept. of Biology, Toledo, Ohio. It has been described in Turgeonet al , PNAS, 73, 3562 (1976) and in Sciaky et al , Plasmid. 1, 238(1978).

Seeds of the inbred yellow Iochief strain of corn were sterilized,germinated, inoculated and further incubated for 7-14 days as describedabove in Example I, parts B and C, except that the corn seedlings wereinoculated with strain T37 rather than strain B6.

At the end of the 7-14 day incubation period, the seedlings were assayedfor enzyme activity as described above in Example II, part C. Using thisprocedure, nopaline production by the cell-free extracts ofT37-inoculated corn seedlings was demonstrated showing that theseedlings were transformed.

EXAMPLE VI

A single colony of the A. tumefaciens strain C58 was inoculated intoYEB, and the bacteria were incubated as described above in Example II,part A. Seeds of the inbred PA91 strain of corn were sterilized,germinated, inoculated and further incubated for 7-14 days as describedabove in Example I, parts B and C, for the inbred yellow Iochief strain.The PA91 strain is a standard inbred strain of corn which iscommercially available. It was obtained from Jean Roberts, Eli Lilly andCo., Greenville, Ind.

At the end of the 7-14 day incubation period, the seedlings were assayedas described above in Example II, part D. As shown in FIG. 7, nopalineproduction by the cell-free extracts was demonstrated showing that thePA91 strain of corn had been transformed. In FIG. 6, lanes 1-10 containthe product produced by incubating the cell-free extracts of singleC-58-inoculated seedlings with nopaline dehydrogenase reaction mediumfor six hours. As can be seen, all ten of the cell-free extractsproduced nopaline showing that the seedlings were transformed.

EXAMPLE VII

A single colony of the A. tumefaciens strain B6 was inoculated into ayeast extract broth, and the bacteria were incubated as described abovein Example I, part A. Seeds of the inbred yellow Iochief strain of cornwere sterilized, germinated, inoculated and further incubated for 7-14days as described above in Example I, parts B and C, except that theseeds were germinated as follows. After being sterilized, the seeds weresoaked for about 12 hours in sterile distilled water. They were thenincubated on sterile moistened Whatman No. 3 paper in sterile Petridishes as described above, but they were only incubated for 1.5 to 2.0days since soaked seeds germinate in a shorter time than do unsoakedseeds.

At the end of the 7-14 day incubation period, the seedlings were assayedfor enzyme activity as described above in Example I, part D. Using thisprocedure, octopine production by cell-free extracts of the infectedseedlings was demonstrated showing that the seedlings were transformed.

EXAMPLE VIII

A single colony of the A. tumefaciens strain LBA 4013 was inoculatedinto YEB, and the bacteria were incubated as described above in ExampleI, part A, for strain B6. The LBA 4013 strain is a mutant strain derivedfrom A. tumefaciens strain Ach5. LBA 4013 contains the wild type Tiplasmid pTiAch5 which is vir⁺, and LBA 4013 codes for the production oflysopine dehydrogenase in suitable plant hosts. LBA 4013 was obtainedfrom Clegg Waldron, Eli Lilly and Co., Indianapolis, Ind. It has beendescribed by Marton et al., in Nature, 277, 129 (1979).

Seeds of the inbred yellow Iochief strain of corn were sterilized,germinated, inoculated and further incubated for 7-14 days as describedabove in Example I, parts B and C, except that the corn seedlings wereinoculated with strain LBA 4013 rather than strain B6.

At the end of the 7-14 day incubation period, the seedlings were assayedfor enzyme activity as described above in Example I, part D. Using thisprocedure, octopine production by cell-free extracts of LBA4013-transformed seedlings was demonstrated showing that the seedlingswere transformed.

EXAMPLE IX A. Preparation of Bacteria

A single colony of the A. tumefaciens strain CA19 was inoculated intoYEB, and the bacteria were incubated as described above in Example I,Part A, for strain B6. The CA19 strain is derived from strain LBA 4013and contains the pTiAch5 plasmid of LBA 4013 which is vir⁺ as describedabove in Example VIII, and strain CA19 codes for the production oflysopine dehydrogenase in suitable plant hosts.

Strain CA19 also contains the micro-Ti plasmid pCEL44. Micro-plasmidpCEL44 comprises a construct consisting of the gene coding forhygromycin phosphotransferase (aphIV) inserted between the 5' promoterand associated amino terminal region-encoding sequence of an octopinesynthase gene and the 3' terminator sequence of a nopaline synthasegene. This construct is assembled between T-DNA border fragments inbroad-host-range vector pKT210. Micro-plasmid pCEL 44 is capable oftransforming plant cells and rendering them resistant to hygromycin.

Strain CA19 is prepared as follows.

1. Culture of Escherichia coli RR1ΔM15/pCEL30 and Isolation of PlasmidpCEL30: Plasmid pCEL30 comprises the right-hand border sequence of theT-DNA and 5' end of the octopine synthase (ocs) gene derived fromplasmid pTiA66. A linker containing a unique Bg1II site is fused in the11th codon of the ocs gene. Attached to the linker are the terminationand polyadenylation signals of the nopaline synthase gene of plasmidpTiC58. Attached to these sequences is a sequence which includes theleft-hand border sequence of the T-DNA derived from plasmid pTiA66. Arestriction site and function map of plasmid pCEL30 is given in FIG. 8.

Plasmid pCEL30 can be conventionally isolated from Escherichia coli K12RR1ΔM15/pCEL30. E. coli RR1ΔM15/pCEL30 is on deposit at the NorthernRegional Research Laboratory (NRRL), Peoria, Ill. 61604, and hasaccession number NRRL B-15915.

The isolation is performed as follows. E. coli RR1ΔM15/pCEL30 is grownin 750 ml of L medium (10 g/l caesin hydrolysate, 5 g/l yeast extract, 5g/l NaCl, 1 g/l glucose, pH 7.4) containing ampicillin at 50 mg/mlaccording to conventional microbiological procedures. The culture isharvested after 24 hours incubation at 37° C. with vigorous shaking.

The culture is centrifuged, and the cell pellet is resuspended in 50 mlfreshly-prepared lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 9 mg/mlglucose, 2 mg/ml lysozyme). After 45 minutes incubation on ice, thesuspension is mixed with 100 ml of a solution that is 0.2N NaOH and 1%SDS. The suspension is then kept on ice for a further 5 minutes. Another90 ml of 3M sodium acetate is added, and the mixture is maintained onice for an additional 60 minutes.

Cell debris is removed by centrifugation, and the supernatant is mixedwith 500 ml ethanol. After 2 hours at -20° C., nucleic acid is pelletedby centrifugation and is resuspended in 90 ml of 10 mM Tris-HCl, pH 8,10 mM EDTA.

The nucleic acid solution is mixed with 90 gm cesium chloride, and 0.9ml of a solution containing 10 mg/ml of ethidium bromide. This mixtureis then centrifuged at 40,000 rpm for 24 hours to purify the plasmidDNA. The plasmid DNA band is recovered and is then recentrifuged at40,000 rpm for 16 hours. The plasmid DNA band is again recovered andfreed of cesium chloride and ethidium bromide by conventionalprocedures. It is next precipitated with 2 volumes of ethanol containing90 g/l ammonium acetate. The pelleted DNA is dissolved in TE buffer (10mM Tris-HCl, pH 8, 1 mM EDTA) at a concentration of 0.2 mg/ml.

2. Culture of E. coli JA221/pOW20 and Isolation of Plasmid pOW20: E.coli JA221/pOW20 is grown as described for E. coli RR1ΔM15/pCEL30 inpart A1 of this example, and plasmid pOW20 is prepared as described forplasmid pCEL30 in part A1of this example.

3. Construction of E. coli RR1ΔM15/pCEL40: Five μg of plasmid pCEL30 DNAare digested with 50 units of Bg1II restriction enzyme in a 150 μlreaction mixture of the composition recommended by the enzymemanufacturer. Restriction and other enzymes can be readily obtained fromthe following sources:

Bethesda Research Laboratories, Inc. Box 6010 Rockville, Md. 20850

Boehringer Mannheim Biochemicals 7941 Castleway Drive P.O. Box 50816Indianapolis, Ind. 46250

New England Bio Labs., Inc. 32 Tozer Road Beverly, Mass. 01915

Digestion is allowed to proceed for 90 minutes at 37° C.

The reaction mixture is first mixed with 8.75 μl of 0.5M Tris-HCl, pH 8,1 mM EDTA and then with 1.25 units of calf intestinal phosphatase (whichcan be purchased from Boehringer Mannheim) and incubated at 37° C. for15 minutes. The mixture is next extracted with buffered phenol, thenwith ether and is precipitated with 2 volumes of ethanol containingammonium acetate. After 30 minutes at -70° C., the DNA is pelleted andredissolved in TE buffer at a concentration of 10 μg/ml.

About 20 μg of plasmid pOW20 DNA is digested with the restrictionenzymes BamHI and Bg1II according to the enzyme manufacturer'srecommended procedures to obtain the aphIV gene. The aphIV gene is an E.coli gene which makes plants containing the gene resistant tohygromycin.

The DNA fragments resulting from this digestion are fractionated byconventional methods of agarose gel electrophoresis and isolated byentrapment on a piece of NA-45 DEAE paper (Schleicher & Schuell Inc.,Keene, N.H. 03431) inserted into the gel during electrophoresis. DNA iseluted from the paper by spinning the paper for 5 seconds with asufficient amount of a high salt buffer (1.0M NaCl; 0.1 mM EDTA; and 20mM Tris, pH 8.0) to cover the paper in a microcentrifuge. The paper isincubated at 55-60° C. for 10-45 minutes with occasional swirling. Thebuffer is removed, and the paper washed with about 50 μl of buffer. TheDNA is extracted first with phenol and then with ether and isresuspended in TE buffer at a concentration of about 25 μg/ml.

Ten ng of the phosphatased, Bg1II-cut plasmid pCEL30 is mixed with 50 ngof the purified ˜1.3 kb BamHI-Bg1II fragment of plasmid pOW20 in a 15 μlligase buffer (50 mM Tris-HCl, pH 7.6; 10 mM MgCl₂ ; 10 mM DTT; and 1 mMATP) containing 0.8 units of T4 DNA ligase (BRL). The mixture isincubated overnight at 15° C.

The ligation mixture is mixed with 15 μl sterile 60 mM CaCl₂ solution.Next, 70 μl of a suspension of competent E. coli RR1ΔM15 cells, whichhas been stored 20× concentrated in 30 mM CaCl₂, 15% glycerol at -70°C., are added. After 60 minutes on ice, the transformation mixture isheat-treated at 42° C. for 2 minutes and is then incubated with 0.5 ml Lmedium for 90 minutes at 37° C.

Samples of the mixture are spread on L medium containing ampicillin at50 mg/l and solidified with agar at 15 g/l. These samples are thenincubated overnight at 37° C. to permit growth of colonies fromtransformed cells.

Colonies resulting from the transformation are inoculated into 5 ml Lmedium containing ampicillin at 50 mg/ml and grown overnight at 37° C.Plasmid DNA is prepared from 1 ml samples of these cultures by theprocedure of Holmes & Quigley, Analytical Biochemistry, 114, 193 (1981)and is redissolved in 50 μl of TE buffer.

4. Construction of Micro-Ti Plasmid pCEL44: Since plasmid pCEL40 is notcapable of replication in Agrobacterium, the micro T-DNA of plasmidpCEL40 was first transferred, as an EcoRI fragment, intobroad-host-range vector pKT210. This broad-host-range vector isavailable from Plasmid Reference Center, Stanford University, Palo Alto,Calif. 94305.

Five μg of plasmid pKT210 are digested with 50 units of EcoRIrestriction enzyme in a 150 μl reaction of a composition recommended bythe enzyme manufacturer. After 90 minutes at 37° C., the reaction istreated with calf intestinal phosphatase as described above in part A3of this example and is dissolved in TE buffer at a concentration of 10μg/ml.

Fifteen μl of a preparation of plasmid pCEL40 DNA, grown as describedabove in part A3 of this example, are digested with 10 units of EcoRIrestriction enzyme in a 20 μl reaction at 37° C. for 90 minutes and arethen extracted with phenol, followed by extraction with ether. Thedigested DNA is precipitated with 2 volumes of ethanol containingammonium acetate at -20° C. and is redissolved in 20 μl TE buffer.

Ten ng of phosphatased, EcoRI-cut pKT210 are ligated with 5 μl ofEcoRI-cut pCEL40 as described above in part A3 of this example, andtransformed into E. coli RR1ΔM15 as described above in part A3 of thisexample.

Transformed cells containing pCEL44 are selected by their ability togrow on solidified L medium containing chloramphenicol at 10 mg/l. Arestriction site and function map of pCEL44 is provided in FIG. 9.

5. Conjugation of pCEL44 Into A. tumefaciens LBA4013 to form StrainCA19: E. coli K12 RR1ΔM15/pCEL44 and E. coli pRK2013 are grown overnightat 37° C. on solidified L medium. A. tumefaciens LBA4013 is grown for 2days at 28° C. on solidified L medium.

One loop of E. coli K12 RR1ΔM15/pCEL44, one loop of E. coli pRK2013 andone loop of A. tumefaciens LBA 4013 are mixed in 1 ml of 30 mM magnesiumsulfate solution. Next, a drop of the mixture is placed on solidified TYmedium (5 g/l caesin hydrolysate, 5 g/l yeast extract, 15 g/l agar) andincubated at 28° C. overnight.

The bacterial growth is resuspended in 3 ml of 10 mM magnesium sulfatesolution and 0.1 ml samples of serial dilutions are spread on solidifiedTY medium containing 100 mg/l nalidixic acid and 2 mg/l chloramphenicoland incubated at 28° C.

Transconjugants give rise to individual colonies after 2 to 4 daysgrowth. These are inoculated singly into 25 ml liquid TY mediumcontaining 100 mg/l nalidixic acid and 2 mg/l chloramphenicol andincubated at 28° C. with shaking for another 2 days. The plasmid contentof the transconjugants is then checked by the method of Casse et al.(Journal of General Microbiology 113:229-242; 1979), and strain CA19containing the wild type pTiAch5 plasmid and the pCEL44 plasmid isisolated.

The CA19 used to practice the method of the present invention wasobtained from Clegg Waldron, Eli Lilly and Co., Indianapolis, Ind. Thepreparation of strain CA19 has also been described in Waldron et al.,Plant Molec. Biol., 5, 103 (1985) which is incorporated herein byreference.

B. Transformation of Corn

Seeds of the inbred yellow Iochief strain of corn were sterilized,germinated, inoculated and further incubated for 7-14 days as describedabove in Example I, parts B and C, except that the corn seedlings wereinoculated with strain CA19 rather than strain B6.

C. Assays

1. Assay of Seedlings for Lysopine Dehydrogenase Activity: At the end ofthe 7-14 day incubation period, the seedlings were assayed for enzymeactivity as described above in Example I, part D. The results are shownin FIG. 6 where lanes 1-10 contain the product produced by incubatingcell-free extracts of single CA19-inoculated seedlings with lysopinedehydrogenase for four hours. As can be seen there, octopine productionin 9 out of 10 cell-free extracts of single CA19-inoculated cornseedlings was demonstrated showing that the seedlings contained lysopinedehydrogenase and were transformed.

2. Assay of Seedlings for Hygromycin Resistance: Some seedlings whichwere inoculated with strain CA19 were incubated only for 3-4 days afterinoculation, at which time they were assayed for resistance tohygromycin as follows. The seedlings were dissected away from theendosperm and scutellum (see FIGS. 1A and 1B) and cut into approximately3 mm cross sections. The cross sections were cultured for three weeks onDuncan's medium (described by Duncan et al. in Planta, 165, 322 (1985))supplemented with 200 ug/ml each carbenicillin (Sigma) and vancomycin(Lilly) or on BN4 medium (Murashige and Skoog major and minor salts(described in Physiol. Plant, 15 473 (1962)), 4 mg/l 2,4-dichlorophenoxyacetic acid as auxin, 9 g/l Difco Bactoagar and 20 g/l sucrose)supplemented with 200 μg/ml each carbenicillin and vancomycin, in thedark, at 25° to 27° C., followed by another three-week passage on thoseantibiotics.

To test the response to hygromycin of the tissue cultures derived fromthe corn seedlings, either whole cross sections plus the tissue whichhas grown up from the cross sections or 100 mg callus samples are placedonto about 50 ml of Duncan's medium or of BN4 medium supplemented withthe aforementioned concentrations of vancomycin and carbenicillin andcontaining about 125 μg/ml of hygromycin B (Lilly) contained in Falcon1005 Petri dishes. This test is read after three weeks of incubation inthe dark at 27° C., by visually checking for growth. Cultures that aregrowing are light in color and show an increase in size. Using thistest, positive growth phenotypes are recovered from cultures derivedfrom CA19-inoculated seedlings showing that the seedlings aretransformed by the heterologous hygromycin gene.

EXAMPLE X A. Preparation of an A. tumefaciens Strain Carrying the GeneConferring Resistant to the Herbicide Glyphosate

1. Culture of E. coli RR1ΔM15/pCEL30 and Isolation of Plasmid pCEL30: E.coli RR1ΔM15/pCEL30 is grown as described above in Example IX, part A1,and plasmid pCEL30 is isolated as described in Example IX, part A1.

2. Bq1II Digestion of Plasmid pCEL30 and Treatment With Calf IntestinalPhosphatase: Five μg of plasmid pCEL30 DNA are digested and treated withcalf intestinal phosphatase as described above in Example IX, part A3.

3. Isolation of Glysophate-Resistant EPSP Sunthase Gene: A gene codingfor a glyphosate-resistant 5-enolphyruvylshikimate 3-phosphate synthasegene (GREPSPS gene) is isolated as described by Comai et al. in Nature,317, 714 (1985) and by Stalker et al., J. Biol. Chem., 260, 4724 (1985)which are incorporated herein by reference. In the final steps of thisprocedure, the GREPSPS gene, in a BamHI fragment cut from plasmidpPMG34, is cloned into plasmid pUC7 to give plasmid pPMG38. The GREPSPSgene is then excised from plasmid pPMG38 as an EcoRI fragment. The EcoRIfragment is then modified using a suitable commercially availablemolecular linker so that it is able to ligate with the unique Bg1II siteon the Bq1II-digested pCEL30 plasmid and so that the EcoRI site isremoved.

The herbicide glyphosate (N-phosphonomethylglycine) is a widely usedbroad-spectrum herbicide that kills both weed and crop species. Itinhibits a metabolic step in the biosynthesis of aromatic compounds, andthe cellular target of glyphosate is 5-enolphyruvyl shikimate3-phosphate synthase (EPSP synthase) which catalyzes the formation of5-enolpyruvylshikimate 3-phosphate from phosphoenolpyuvate andshikimate, and inhibition of this step of the shikimate pathwayeventually leads to cellular death. The GREPSPS gene is a mutant alleleof the aroA locus of Salmonella typhimurium which encodes a EPSPsynthase in which the substitution of a serine for proline causes adecreased affinity of the enzyme for glyphosate.

4. Ligation: Ten ng of the phosphatased, Bg1II-cut plasmid pCEL30, asprepared above in Example IX, part A3, are mixed with 50 ng of theGREPSPS gene (including the attached linker) in a 15 μl ligase buffercontaining 0.8 units of T4 DNA ligase. The mixture is incubatedovernight at 15° C. The ligation mixture is used to transform competentE. coli RR1ΔM15 cells as described in Example IX, part A3.

5. Construction of Micro-Ti Plasmid Carrying the GREPSPS Gene: Afterselection of transformed cells produced as described in part A4 of thisexample on L medium containing ampicillin as described above in ExampleIX, part A3, the plasmids from the transformed E. coli RR1ΔM15 cells aretransferred as described above in Example IX, part A4, as an EcoRIfragment, into broad- host-range vector pKT210.

6. Conjugation Into A. tumefaciens LBA 4013: The plasmid carrying theGREPSPS gene on broad-host-range vector pKT210 is transferred into A.tumefaciens strain LBA 4013 by conjugation as described above in ExampleIX, part A5. The resulting transconjugants are selected as describedabove in Example IX, part A5, and a new strain of A. tumefacienscarrying the GREPSPS gene which is herein referred to as strain LBA4013/GREPSPS, is isolated.

B. Transformation of Corn

Seeds of the inbred yellow Iochief strain of corn are sterilized,germinated, inoculated and further incubated as described above inExample I, parts B and C, except that the corn seedlings were inoculatedwith strain LBA 4013/GREPSPS rather than strain B6. After 7 days ofincubation, the infected seedlings are planted in pots in potting soil.

C. Assays

1. Assay For Enzymatic Activity in Pollen: Sixty days after the plantingof the seedlings, samples of the pollen of five plants are individuallyassayed for the presence of lysopine dehydrogenase as described above inExample III, part B. The results of the electrophoresis of the productsproduced by incubating the cell-free extracts of the pollen of the fiveplants show that three out of five of the cell-free extracts of thepollen produce octopine showing that the pollen is transformed.

2. Assay for EPSP Synthase Activity: Leaves derived from the meristemare assayed seven weeks after planting of the seedlings for EPSPsynthase activity according to the method of Boocock and Coggins, FEBSLetters, 154, 127 (1983) which is incorporated herein by reference. Fiveleaves from five separate plants are assayed, and three are found tocontain EPSP synthase activity showing that transformation had occurred.

3. Assay For Resistance To Glyphosate: The three plants found to containEPSP synthase activity in their leaves as a result of the assay in partC2 of this example are sprayed with the equivalent of 0.5 kg/hectare ofthe isopropylamine salt of glyphosate. All three plants showconsiderable tolerance to glyphosate as compared to controls.

None of the foregoing description of the preferred embodiments isintended in any way to limit the scope of the invention which is setforth in the following claims. Those skilled in the art will recognizethat many modifications, variations and adaptations are possible.

EXAMPLE XI A. Transformation of Corn

Strain CA19 was cultured as described in Example IX, part A, and yellowIochief corn was sterilized, germinated, inoculated with CA19 andinoculated as described in Example I, parts B and C. Seedlings were alsoinoculated with A. tumefaciens strain CA17 which is identical to strainCA19, except that the gene that codes for hygromycin resistance isinserted in the antisense direction. Finally, seedlings were alsoinoculated with YEB alone. After seven days of incubation, all of theinoculated seedlings were planted.

B. Assays

1. Assay For Enzymatic Activity in the Upper Leaves of TransformedPlants: As the plants reached sexual maturity, their upper leaves wereassayed for the presence of lysopine dehydrogenase activity as describedin Example III, part B2, except that lysopine dehydrogenase reactionmedium was used. Leaves of five plants derived from seedlings inoculatedwith CA19, of two plants derived from seedlings inoculated with CA17 andof three plants inoculated with YEB were assayed.

The results are shown in FIG. 14. In FIG. 14, the lanes contain thefollowing materials:

    ______________________________________                                        CONTENTS OF LANE                                                                              Proculum   Plant                                                    Plant     Used on    Part                                               Lane  Code No.  Seedling   Sampled  Comments                                  ______________________________________                                        1     --        --         --       Synthetic                                                                     octopine                                                                      standard                                  2     S-1       YEB        Flag leaf                                                                              --                                        3     19-6      CA19       Leaf below                                                                             --                                                                   flag leaf                                          4     19-6      CA19       Tiller   --                                        5     17-4      CA17       Ear shoot                                                                              --                                        6     19-3      CA19       Ear shoot                                                                              --                                        7     19-3      CA19       Leaf below                                                                             --                                                                   flag leaf                                          8     S-5       YEB        Leaf below                                                                             --                                                                   flag leaf                                          9     17-3      CA17       Leaf below                                                                             --                                                                   flag leaf                                          10    17-3      CA17       Second leaf                                                                            --                                                                   below flag                                                                    leaf                                               11    19-4      CA19       Leaf below                                                                             --                                                                   flag leaf                                          12    S-8       YEB        Leaf below                                                                             --                                                                   flag leaf                                          13    19-5      CA19       Flag leaf                                                                              --                                        14    19-5      CA19       Leaf below                                                                             --                                                                   flag leaf                                          15    19-2      CA19       Flag leaf                                                                              --                                        16    19-2      CA19       Leaf below                                                                             --                                                                   flag leaf                                          ______________________________________                                    

As shown in FIG. 14, none of the extracts of leaves derived fromYEB-inoculated seedlings produced octopine, whereas 8 out of 12 extractsof leaves derived from CA19-inoculated and CA-17-inoculated seedlingsproduced octopine.

2. Assay For Bacteria In Upper Leaves of Transformed Plants: Aliquots ofthe extracts of the leaves used in the lysopine dehydrogenase assaydescribed in part B2 of this example were also plated to determine ifany bacteria were present in these extracts. Any bacterial coloniesgrowing up as a result of these platings were transferred to alactose-containing medium diagnostic for Agrobacteruim. None of thebacteria that grew up as a result of the original platings (either inextracts of leaves derived from YEB-inoculated seedlings, fromCA19-inoculated seedlings or CA17-inoculated seedlings) oxidized lactoseto lactic acid showing that none of them were Agrobacterium of the typeused to inoculate the seedlings.

3. Assay For Enzymatic Activity in Leaves of F₁ Plants: Using theresults of the above assay, plants 19-5 and 19-3 were chosen for furtherstudy. Plant 19-5 was chosen because extracts of both of the two topleaves were positive for lysopine dehydrogenase activity indicating thatthis plant might have a transformed sector extending into the tassel.Plant 19-3 was chosen because extracts of its ear shoot and leaf belowthe flag leaf were positive for lysopine dehydrogenase activityindicating that this plant might have a transformed sector extendinginto the ear and a transformed sector extending into the tassel.

These two plants were self pollinated. Then, 26-27 days postpollination, the immature progeny ears of plants 19-3 and 19-5 wereremoved from the plants and surface sterilized. The late maturityembryos of the ears from these plants were excised and planted onhalf-strength Murashige and Skoog medium. The embryos were allowed togerminate sterilely in the light for 8-10 days, at which time they wereplanted in soil.

The meristem-derived leaves of the F₁ seedlings which survivedtransplanting to soil were assayed for lysopine dehydrogenase activityas described in Example III, part B2, except that lysopine dehydrogenasereaction medium was used. The intensity of the staining of the spots onthe electrophoretogram that co-migrated with octopine was rated. Theresults are presented in Table 1. As shown there, some of the leaves ofthe seedlings produced octopine, showing the sexual transmission of thistrait to the F₁ generation.

    ______________________________________                                                 Leaves From    Leaves From                                                    Plants Produced                                                                              Plants Produced                                                By Embryos Taken                                                                             By Embryos Taken                                      Rating   From Plant 19-3                                                                              From Plant 19-5                                       ______________________________________                                        Dead     85              9                                                    (no test)                                                                     Negative 71             76                                                             (25 subsequently died)                                                                       (7 subsequently died)                                 Weak      5             17                                                    Positive  (3 subsequently died)                                                                       (0 subsequently died)                                 Positive 34             27                                                              (8 subsequently died)                                                                       (3 subsequently died)                                 Strong    3              3                                                    Positive  (0 subsequently died)                                                                       (0 subsequently died)                                 ______________________________________                                    

TRANSFORMATION OF OTHER SPECIES OF GRAMINEAE EXAMPLE XII

A single colony of the A. tumefaciens strain B6 was inoculated into ayeast extract broth, and the bacteria were incubated as described abovein Example I, part A. Seeds of rye were sterilized, germinated,inoculated and further incubated for 7-14 days as described above inExample I, parts B and C. The seedlings were inoculated in the apicalmeristem, an area of rapidly dividing cells that gives rise to the germline cells.

At the end of the 7-14-day incubation period, the seedlings were assayedfor enzyme activity as described above in Example I, part D. The resultsof the electrophoresis of the products produced by adding the cell-freeextracts of the B6-inoculated rye seedlings to lysopine dehydrogenasereaction medium are shown in FIG. 10. In that figure, lane 1 containsthe synthetic octopine standard and lanes 2-6 contain the productproduced by incubating the cell-free extract of single B6-inoculated ryeseedlings with lysopine dehydrogenase reaction medium. The results shownin FIG. 10 demonstrate that octopine production is caused by thecell-free extracts of three out of four B-6-inoculated rye seedlingstested (lanes 2, 3, 4 and 6). These results show that the rye seedlingshave been transformed by infection with the vir⁺ A. tumefaciens strainB6.

EXAMPLE XIII

A single colony of the A. tumefaciens strain B6 was inoculated into ayeast extract broth, and the bacteria were incubated as described abovein Example I, part A. Barley seeds were sterilized, germinated,inoculated and further incubated for 7-14 days as described above inExample I, parts B and C. The seedlings were inoculated in the apicalmeristem, an area of rapidly dividing cells that gives rise to the germline cells.

At the end of the 7-14-day incubation period, the seedlings were assayedfor enzyme activity as described above in Example I, part D. The resultsof the electrophoresis of the products produced by adding the cell-freeextracts of B6-inoculated barley seedlings to lysopine dehydrogenasereaction medium are shown in FIG. 11. In that figure, lane 1 containsthe synthetic octopine standard and lanes 2-6 contain the productproduced by incubating the cell-free extract of single B6-inoculatedbarley seedlings with lysopine dehydrogenase reaction medium. Theresults shown in FIG. 11 demonstrate that octopine production is causedby five out of five cell-free extracts of B6-inoculated barley seedlingstested and show that the seedlings have been transformed.

EXAMPLE XIV

A single colony of the A. tumefaciens strain C58 was inoculated intoYEB, and the bacteria were incubated as described above in Example II,part A. Oat seeds were sterilized, germinated, inoculated and furtherincubated for 7-14 days as described above in Example I, parts B and C.The seedlings were inoculated in the apical meristem, an area of rapidlydividing cells that gives rise to the germ line cells.

At the end of the 7-14-day incubation period, the seedlings were assayedas described above in Example II, part D. The results are shown in FIG.12. In FIG. 12, lane 1 contains nopaline dehydrogenase reaction mediumalone, lane 2 contains the nopaline standard, lanes 3-11 contain theproduct produced by incubating the cell-free extracts of singleC58-inoculated oat seedlings with nopaline dehydrogenase reaction mediumand lane 12 contains synthetic octopine. As can be seen, six out of nineof the cell-free extracts of single C53-inoculated oat seedlingsproduced nopaline showing that the seedlings were transformed.

EXAMPLE XV

A single colony of the A. tumefaciens strain C58 was inoculated intoYEB, and the bacteria were incubated as described above in Example II,part A. Wheat seeds were sterilized, germinated, inoculated and furtherincubated for 7-14 days as described above in Example I, parts B and C.The seedlings were inoculated in the apical meristem, an area of rapidlydividing cells that gives rise to the germ line cells.

At the end of the 7-14-day incubation period, the seedlings were assayedas described above in Example II, part D. The results are shown in FIG.13. In FIG. 13, lane 1 contains the nopaline standard, lanes 2-6 containthe product produced by incubating the cell-free extracts of singleC58-inoculated wheat seedlings with nopaline dehydrogenase reactionmedium, lane 7 contains synthetic octopine, lane 8 contains a mixture ofsynthetic octopine and lysopine dehydrogenase reaction medium and lanes9-15 contain the product produced by incubating the cell-free extractsof single C58-inoculated wheat seedlings with lysopine dehydrogenasereaction medium. As can be seen, three out of five of the cell-freeextracts of the C58-inoculated wheat seedlings produced nopaline whenincubated with nopaline dehydrogenase reaction medium, showing that theseedlings were transformed. None of the cell-free extracts producedoctopine when incubated with lysopine dehydrogenase reaction medium.

We claim:
 1. A method of producing transformed Gramineae, said methodcomprising:making a wound in a graminaceous seedling with newly emergedradicle and stem, said wound being made in an area of the seedlingcontaining rapidly dividing cells, wherein said area extends from thebase of the scutellar node to slightly beyond the coleoptile node; andinoculating the wound with vir+ Agrobacterium tumefaciens.
 2. The methodof claim 1 wherein about four wounds are made in the seedling, and atotal of about 10⁸ Agrobacterium tumefaciens cells are used to inoculatethe wounds.
 3. The method of claim 1 wherein the vir⁺ Agrobacteriumtumefaciens contains a vector comprising genetically-engineered T-DNA.4. The method of claim 1 or 3 wherein the Gramineae is selected from thegroup consisting of corn, wheat, rye, barley and oats.